Stabilized albumin-free recombinant factor VIII preparation having a low sugar content

ABSTRACT

A stable albumin-free Recombinant Factor VIII (rFVIII) formulation in lyophilized form having both crystalline and amorphous components and comprising, when reconstituted with water, about 65 to 400 mM glycine, up to 50 mM histidine, 15 to 60 mM sucrose, up to 50 mM NaCl, up to 5 mM CaCl 2  and 50 to 1500 IU/ml of rFVIII. A very preferred formulation comprises upon reconstitution with water about 290 mM glycine, 20 mM histidine, 30 mM sucrose, 30 mM NaCl, 2.5 mM CaCl 2  and 50 to 1500 IU/ml of rFVIII. The residual water content of the lyophilized preparation is about 1 to 3% by weight, preferably about 1% by weight.

BACKGROUND OF THE INVENTION

1. Field

This application relates generally to pharmaceutical formulations andparticularly to a lyophilized formulation for rFVIII which is stabilizedwithout albumin (albumin-free).

2. Prior Art

Factor VIII is a well known plasma protein that is essential to theblood clotting process. Although Factor VIII can be and currently isobtained from human plasma, efforts have been made in recent years tomanufacture Factor VIII from recombinant sources (rFVIII) to avoidpotential contaminations associated with blood products. In addition, arecombinant source for Factor VIII provides a virtually unlimited supplyof the coagulation factor, thus avoiding supply limitations associatedwith using donated blood plasma as a source material.

Since one of the advantages of a rFVIII product is that it is notderived from human plasma and thus avoids potential contamination from ahuman plasma source, it has been a goal in rFVIII manufacturing todevelop a stable formulation for rFVIII which can be described as beingentirely free of any human source raw materials. Unfortunately, however,rFVIII is a labile protein and, like many other therapeutic proteins, itcan become unstable during storage. To overcome such instability, it hasbeen common practice to include human serum albumin to the product as astabilizer. Albumin has been found to be a good stabilizer of FVIII andis used in numerous commercial products on market. By using humanalbumin as a rFVIII stabilizer, however, one of the advantages of havinga recombinant product in the first place (i.e., avoiding any human basedsource materials) is lost.

There have been some recently described albumin-free formulations forFactor VIII in both low and high ionic strength media using sodiumchloride, calcium chloride and histidine as a buffer ion. In addition,basic amino acids such as lysine and sugars such as mannitol, sucrose ormaltose have been used. To achieve stability in an albumin-free FactorVIII formulation while retaining necessary isotonicity needed fortherapeutic use, approximately 10% sugar has been used in suchalbumin-free formulations. See for example U.S. Pat. No. 4,877,608 (lowionic strength formulation). European patent 0 314 095 discloses anotheralbumin-free formulation having a high ionic strength and histidine as abuffering agent. The '608 patent is concerned with a liquid solution andnot a lyophilized product that must be able to undergo freeze dryingcycles necessary to prepare the product. European patent 314 095includes a relatively high amount of sodium chloride and is primarilyused in a liquid formulation.

Other patents concerned with Factor Vlll formulations include U.S. Pat.No. 5,399,670, which describes the use of arginine in an albumincontaining freeze dried formulation. See also WO 95/01804 whichdescribes a formulation that includes no sucrose or glycine and U.S.Pat. No. 4,440,679 and U.S. Pat. No. 4,623,717 (both to Fernandes etal.) showing the use of at least 30% by weight sugars in combinationwith amino acids to stabilize FVIII in the liquid state underpasteurization conditions (60° C., at least 10 hours).

In addition to the above Factor VIII formulations there are severalother patents related to the purification and/or stabilization of FactorVIII. These include U.S. Pat. No. 5,288,853 which covers a multi-stepmanufacturing process including the use of a heparin-coupled columnfollowed by the addition of glycine to form a purified Factor VIIIproduct.

U.S. Pat. No. 5,399,670 covers a process for producing a lyophilizedFactor VIII preparation of enhanced solubility which requires additionof arginine to the Factor VIII solution prior to lyophilization.

U.S. Pat. No. 5,259,951 covers a multi-step method of purifying FactorVIII from plasma using ion exchange columns.

U.S. Pat. No. 4,758,657 covers a multi-step process for separatingFactor VIII:C from plasma in which at least one of the steps requiresthe adsorption of Factor VIII:C on a hydrophobic interaction matrix.

In addition to the above patents, there is a very recently describedformulation for Factor lX (FIX) which appears similar to the formulationfor rFVIII disclosed herein. See Abstract 244 by L. Bush et al.,Hemophilia, Vol. 2, Supplement 1, p. 64 (June, 1996). FIX is apro-enzyme that is converted to an active proteolytic enzyme. FVIIIserves, on the other hand, as a co-factor along with other coagulationcomponents in effecting blood coagulation. The molecular weight of FVIIIis about 340,000 Daltons whereas FIX has a molecular weight of about56,000 to 57,000. FVIII is very sensitive to proteolytic processing withconcomitant loss of coagulant activity. It is well known that FactorVIII is inherently more unstable than FIX and freeze dried concentratesof each factor demonstrate marked differences in stability on storage atvarious temperatures. Unlike FVIII, FIX includes unique gammacarboxylation of 12N terminal glutamic acid residues, thus providing apossible basis for differential stability. Thus a formulation for FIXwould not necessarily suggest a formulation for FVIII.

In Pharmaceutical Research, Volume 12, No. 6, pages 831-837, 1995, thereis also disclosed a formulation for stabilized recombinant humaninterleukin-1 receptor antagonist similar to that disclosed below.

Despite the past and recent efforts to develop a stable rFVIIIpreparation that can be successfully lyophilized and later reconstitutedrapidly in water, to date it has been difficult to provide a formulationthat not only avoids the use of human products such as albumin but alsomeets the requirements for proper lyophilization and rapidreconstitution and isotonicity while at the same time providing a rFVIIIhaving long term stability, with a pharmaceutically acceptableshelf-life.

To our surprise we have now found that such a preparation is possible.In the course of developing this formulation, we found that histidinewhich has been taught and used in the prior art as a buffering agent,actually had a de-stabilizing effect on Iyophilized albumin-freeformulations. We have found however that the de-stabilizing effects ofhistidine can be effectively overcome by a novel formulation of salts,glycine and sucrose, the combination of which was found to have abeneficial effect in stabilizing rFVIII. This mixture also protects therFVIII across multiple freeze thaw cycles during the lyophilizationprocess, and it provides rapid reconstitution of the lyophilized productwith water. The formulation includes both crystalline and amorphouscomponents, unlike most prior art formulations which are essentiallyamorphous. The formulation of our invention remains stable in the liquidstate for at least twenty-four hours at room temperature. Details of ourformulation and its use are described below.

SUMMARY OF THE INVENTION

Our improved rFVIII formulation is a pharmaceutically acceptablealbumin-free lyophilized product which can be rapidly reconstituted inwater (within 30 seconds) and is suitable for treating hemophilia. Thelyophilized preparation comprises a novel mixture of salts, amino acidsand sucrose. The product is stable at room temperature and, unlike theprior art the formulations, comprises a relatively low level of sugar.

The formulation comprises, when reconstituted with water, the followingingredients:

glycine about 65 to 400 mM, preferably 290 mM,

histidine up to about 50 mM, preferably 1 mM to 50 mM, very preferably20 mM,

sucrose about 15 to 60 mM, preferably 30 mM,

NaCl up to about 50 mM, preferably 1 mM to 50 mM, very preferably 30 mM,

CaCl₂ up to about 5 mM, preferably 0.1 to 5 mM, very preferably 2.5 mM,and

rFVIII about 50 to 1500 lU/ml.

In a preferred lyophilized formulation, the amount of residual watershould be about 1 to 3% by weight, preferably about 1% by weight.

BRIEF DESCRIPTION OF THE FIGURE

The figure is a graph comparing the potency over time at 40° C. of theformulation of this disclosure having a relatively low sugar content(upper curve) with a formulation taught by the prior art having arelatively high sugar content (lower curve).

DETAILED DESCRIPTION OF THE INVENTION

The objective that led to this invention was to identify an albumin-freeformulation that offered stability to rFVIII (minimal or less than about20% loss in potency) across various process steps such asultrafiltration/diafiltration, storage of frozen bulk, freeze-thaweffects and lyophilization. In addition, a fast dissolving product wasdesired with stability in the reconstituted liquid state. Finally, apharmaceutically acceptable lyophilized product with an appropriateshelf-life was desired which could be lyophilized with a shortfreeze-drying cycle.

Proteins do not crystallize during lyophilization. The goal of a dryingprocess should be to convert the aqueous protein solution into anamorphous phase to protect the proteins from chemical and/orconformational instability caused by a crystalline (or total lack ofwater) environment. Thus, it is common to include significant amounts ofalbumin (up to 1%) to provide an amorphous phase (component) tostabilize the proteins.

Based on the overall objectives, a formulation with both a crystallinecomponent to allow rapid lyophilization and an amorphous component tostabilize the rFVIII was developed. As used herein the expressioncrystalline with an amorphous component means that the formulationcomprises two or more distinct phases at least one of which iscrystalline and one of which is amorphous. Solids can exist incrystalline or amorphous form. Crystalline materials are characterizedas having defined structure, stoichiometric compositions, and meltingpoints. By contrast, amorphous materials have no clearly definedmolecular structure and can be described as a super-cooled liquid withan extremely high viscosity such as a viscoelastic "rubber" or a morerigid brittle glass. It is thought that other sugars such as maltose,trehalose, and maltotriose may be included to contribute to theamorphous component. Mannitol may be included to contribute to thecrystalline component of the formulation.

The strategy employed to identify a pharmaceutically-acceptablealbumin-free formulation of rFVIII was as follows:

(a) The starting material was highly purified rFVIII that was purifiedusing orthogonal chromatographies. These are defined as chromatographicprocesses which operate under distinct modes and principles and aretypically used in succession. As a result, the protein can be rapidlypurified through application of different more effective purificationmethods. This resulted in Factor VIII that was at least 90% pure (by gelelectrophoresis) with specific activities greater than 2000 lU/mgprotein. Theoretical purity of rFVIII has been a subject of controversybut is thought to be about 3500-5000 lU/mg protein.

(b) The protein was formulated by ultrafiltration/diafiltration (UF/DF)and investigated for recovery across UF/DF, susceptibility tofreeze-thaw, and liquid stability under different incubationtemperatures.

(c) Potential formulations were further characterized for their thermalbehavior by DSC (differential scanning calorimetry). Glass transitiontemperatures (Tg'), devitrification temperature (Td') and eutecticmelting temperature (Te') were determined. This information was used toidentify formulations that could be rapidly lyophilized and weretargeted for further investigation.

(d) The lead formulations were lyophilized using a rapid freeze dryingcycle, and stability analyses were done under standard and acceleratedstorage temperatures.

(e) Stable formulations were readily identified from samples stored at40° C. for various time points.

In analyzing the results of numerous studies that led to the formulationof this invention, a multi-variable experimental design strategy andprogram was used to screen a panel of ingredients that was comprised ofmixtures of amino acids salts and sugars. The results were analyzedusing a sophisticated program to resolve any interactions between theingredients, and a multi-variable response-surface analysis of the datawas generated. To our surprise, it was found that histidine (commonlyused the prior art) actually had a de-stabilizing effect on rFVIIIformulations. This led to the need to critically examine the criteriafor the various ingredients in the formulation we found was finallyacceptable.

EXAMPLE 1

The effect on stability of the lyophilized rFVIII was investigated bytitrating various amounts of histidine in a rFVIII mixture comprising150 mM NaCl, 2.5 mM CaCl₂ and 165 mM mannitol. The results are shownbelow.

                  TABLE                                                           ______________________________________                                        Percent of initial potency after two weeks/40° C. storage in the       presence                                                                      of histidine.                                                                              % of Initial                                                     Histidine (mM)                                                                             Activity at 2 weeks/40° C.                                ______________________________________                                        20           5.9%                                                             55           6.3%                                                             75           2.5%                                                             100          1.9%                                                             ______________________________________                                    

As can be seen from the above data, increasing amounts of histidineresulted in decreased potency of reconstituted lyophilized rFVIII in adose-dependent fashion. This result suggests that histidine does notplay a role in stabilization of FVIII in the lyophilized state.

EXAMPLE 2

Stability of rFVIII in high and low sugar formulations.

Recombinant Factor VIII was prepared in two formulations. Instabilitywas investigated under accelerated storage conditions of 40° C.

The high sugar formulation, similar to that of the prior art, was anamorphous formulation containing, on reconstitution with water, 50 mM ofsodium chloride, 2.5 mM of calcium chloride, 5 mM of histidine, and 10%by wt maltose.

The low sugar containing formulation of this disclosure was crystallinewith an amorphous component of 1% sucrose (30 mM sucrose) to stabilizethe protein. This formulation, on reconstitution with WFI, contained 30mM of sodium chloride, 2.5 mM of calcium chloride, 20 mM of histidine,290 mM glycine and approximately 200 IU/mi of rFVIII. This formulationis compared with the prior art formulation in the figure where it can beseen that the low sugar rFVIII formulation of this disclosure isconsiderably more stable over time than the high sugar stabilizedproduct of the prior art.

Given the above disclosure, it is thought that numerous variations willoccur to one skilled in the art. Therefore, it is intended that theabove examples should be construed as illustrative only and that thescope of this invention should be limited only by the following claims.

I claim:
 1. A stable, albumin-free, lyophilized rFVIII preparationcomprising, when reconstituted in water, about65 to 400 mM glycine, upto 50 mM histidine, 15 to 60 mM sucrose, up to 50 mM NaCl, up to 5 mMCaCl₂, and 50 to 1500 IU rFVIII/ml.
 2. A stable, albumin-freelyophilized rFVIII preparation comprising, when reconstituted withwater, about290 mM glycine, 20 mM histidine, 30 mM sucrose, 30 mM NaCl,2.5 mM CaCl₂, and 50 to 1500 IU rFVIII/ml.
 3. The lyophilizedpreparation of claim 1 wherein the residual water content is about 1 to3% by weight.
 4. The lyophilized preparation of claim 2 wherein theresidual water content is about 1% by weight.
 5. A stable, albumin-free,lyophilized rFVIII preparation comprising, when reconstituted in water,about65 to 400 mM glycine, up to 50 mM histidine, 15 to 60 mM sucrose,up to 50 mM NaCl, up to 5 mM CaCl₂, and 50 to 1500 IU rFVIII/mlthepreparation being crystalline with an amorphous component and includinga residual water content of about 1 to 3% by weight and having theproperty of being rapidly reconstituted in water.
 6. The product ofclaim 5 wherein the preparation reconstituted in water within 30seconds.